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Novel Mechanism of Inhibition by the P2 Receptor Antagonist PPADS of ATP-Activated Current in Dorsal Root Ganglion Neurons

Novel Mechanism of Inhibition by the P2 Receptor Antagonist PPADS of ATP-Activated Current in... Abstract The antagonist pyridoxal-phosphate-6-azophenyl-2′,4′-disulfonic acid (PPADS) has been proposed to selectively antagonize the actions of ATP at P2X receptors. Whole cell patch-clamp recording techniques therefore were used to characterize PPADS inhibition of ATP-activated current in bullfrog dorsal root ganglion (DRG) neurons. PPADS, 0.5–10 μM, inhibited ATP-activated current in a concentration-dependent manner with an IC 50 of 2.5 ± 0.03 μM. PPADS produced a gradual decline of ATP-activated current to a steady state, but this was not an indication of use dependence as the gradual declining component could be eliminated by exposure to PPADS before ATP application. In addition, ATP-activated current recovered completely from inhibition by PPADS in the absence of agonist. The slow onset of inhibition by PPADS was not apparently due to an action at an intracellular site as inclusion of 10 μM PPADS in the recording pipette neither affected the ATP response nor did it alter inhibition of the ATP response when 2.5 μM PPADS was applied externally. PPADS, 2.5 μM, decreased the maximal response to ATP by 51% without changing its EC 50 . PPADS inhibition of ATP-activated current was independent of membrane potential between −80 and +40 mV and did not involve a shift in the reversal potential of the current. The magnitude of PPADS inhibition of ATP-activated current was dependent on the duration of the prior exposure to PPADS. The time constants of both onset and offset of PPADS inhibition of ATP-activated current did not differ significantly with changes in ATP concentration from 1 to 5 μM. Recovery of ATP-activated current from PPADS inhibition also exhibited a slow phase that was not accelerated by the presence of agonist and was dependent on the concentration of PPADS. The apparent dissociation rate of PPADS from unliganded ATP-gated ion channels was much greater than the rate of the slow phase of recovery of ATP-activated current from PPADS inhibition. The results suggest that PPADS can inhibit P2X receptor function in a complex noncompetitive manner. PPADS produces a long-lasting inhibition that does not appear to result from open channel block but rather from an action at an allosteric site apparently accessible from the extracellular environment that involves a greatly reduced rate of dissociation from liganded versus unliganded ATP-gated ion channels. Footnotes Present address and address for reprint requests: Dept. of Lead Discovery, AstraZeneca, R and D Boston, Three Biotech, One Innovation Dr., Worcester, MA 01605. The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked “ advertisement ” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Copyright © 2000 The American Physiological Society http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png Journal of Neurophysiology The American Physiological Society

Novel Mechanism of Inhibition by the P2 Receptor Antagonist PPADS of ATP-Activated Current in Dorsal Root Ganglion Neurons

Journal of Neurophysiology , Volume 83 (5): 2533 – May 1, 2000

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Publisher
The American Physiological Society
Copyright
Copyright © 2011 the American Physiological Society
ISSN
0022-3077
eISSN
1522-1598
Publisher site
See Article on Publisher Site

Abstract

Abstract The antagonist pyridoxal-phosphate-6-azophenyl-2′,4′-disulfonic acid (PPADS) has been proposed to selectively antagonize the actions of ATP at P2X receptors. Whole cell patch-clamp recording techniques therefore were used to characterize PPADS inhibition of ATP-activated current in bullfrog dorsal root ganglion (DRG) neurons. PPADS, 0.5–10 μM, inhibited ATP-activated current in a concentration-dependent manner with an IC 50 of 2.5 ± 0.03 μM. PPADS produced a gradual decline of ATP-activated current to a steady state, but this was not an indication of use dependence as the gradual declining component could be eliminated by exposure to PPADS before ATP application. In addition, ATP-activated current recovered completely from inhibition by PPADS in the absence of agonist. The slow onset of inhibition by PPADS was not apparently due to an action at an intracellular site as inclusion of 10 μM PPADS in the recording pipette neither affected the ATP response nor did it alter inhibition of the ATP response when 2.5 μM PPADS was applied externally. PPADS, 2.5 μM, decreased the maximal response to ATP by 51% without changing its EC 50 . PPADS inhibition of ATP-activated current was independent of membrane potential between −80 and +40 mV and did not involve a shift in the reversal potential of the current. The magnitude of PPADS inhibition of ATP-activated current was dependent on the duration of the prior exposure to PPADS. The time constants of both onset and offset of PPADS inhibition of ATP-activated current did not differ significantly with changes in ATP concentration from 1 to 5 μM. Recovery of ATP-activated current from PPADS inhibition also exhibited a slow phase that was not accelerated by the presence of agonist and was dependent on the concentration of PPADS. The apparent dissociation rate of PPADS from unliganded ATP-gated ion channels was much greater than the rate of the slow phase of recovery of ATP-activated current from PPADS inhibition. The results suggest that PPADS can inhibit P2X receptor function in a complex noncompetitive manner. PPADS produces a long-lasting inhibition that does not appear to result from open channel block but rather from an action at an allosteric site apparently accessible from the extracellular environment that involves a greatly reduced rate of dissociation from liganded versus unliganded ATP-gated ion channels. Footnotes Present address and address for reprint requests: Dept. of Lead Discovery, AstraZeneca, R and D Boston, Three Biotech, One Innovation Dr., Worcester, MA 01605. The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked “ advertisement ” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Copyright © 2000 The American Physiological Society

Journal

Journal of NeurophysiologyThe American Physiological Society

Published: May 1, 2000

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